1. Field of the Invention
The present invention relates to a microbially produced peptide having essentially the amino acid sequence of human pancreactic secretory trypsin inhibitor (h-PSTI). The present invention further relates to variants of such peptide wherein one or more of the amino acids in the original sequence are replaced by other amino acids. These peptides show an advantageously modified specificity in their inhibitory action. A method of preparation of the peptides and their pharmaceutical use is also described.
2. Background Information
The lysosomal elastase (leukocyte elastase); J. G. Bieth (1986) p. 217-320 (in Regulation of Matrix Accumulation, Mecham ed., Academic Press, Orlando) from polymorphonuclear granulocytes is a potent intracellular protease which is stored in lysosomes and fulfills its physiological function, the intracellular protein breakdown, in phagolysosomes. The major functional role of lysosomal proteases (elastase, cathepsin G, etc.; H. Fritz et al (1984) in Selected Topics in Clinical Enzymology, Goldberg and Werner ed., Walter de Gruyter, Berlin vol. 2, p. 305-328) is the degradation of phagocytized material from either the organism itself (e.g., metabolic products, injured tissue) or from invasive organisms (bacteria, viruses, molds, etc.).
Upon release into the extracellular space (blood or interstitial fluid) elastase is rapidly bound by potent endogenous inhibitors such as .alpha..sub.1 -PI (.alpha..sub.1 -protease inhibitor; J. Travis and G. S. Salvesen, (1983), Ann. Rev. Biochem, p. 655-709,) in plasma, and/or antileukoprotease (also called HUSI-I, human seminal proteinase inhibitor); H. Schiessler et al, (1978), p. 195-207, in Neutral Proteases of Human Polymorphonuclear Leukocytes, Havemann and Janoff ed., Urban & Schwarzenberg, Baltimore) in mucous secretions.
Due to a hereditary .alpha..sub.1 -PI-deficiency (J. B. Bieth, 1986) or as a consequence of massive extracellular release of elastase (in acute and chronic inflammations, polytrauma or shock; H. Fritz et al, 1984) the protection of the organism against the degradative potential of elastase by natural protease inhibitors is insufficient. An excessive and locally even total consumption of the endogenous protease inhibitors is caused by (i) complex formation with elastase, (ii) proteolytic inactivation by various lysosomal proteases, and (iii) particularly by oxidative inactivation (.alpha..sub.1 -PI) (J. B. Bieth (1986); H. Fritz et al. (1984); J. Travis and G. S. Salvesen (1983); see above).
The consequence is an extensive proteolytic degradation of connective tissues, as well as of humoral proteins including coagulation-, fibrinolysis-, and complement factors by elastase and other lysosomal proteases (e.g., cathepsin G) leading to severe clinical symptoms like emphysema, shock lung, adult respiratory distress syndrome, coagulation disorders, kidney and liver failure, etc. (additionally to the above references: Neue Wege in der Entzundungsdiagnostik, PMN Elastase; M. Jochum et al (ed), (1985), GIT Verlag, Darmstadt; C. T. Lee et al, (1981) N. Engl. J. Med., 304, 192-196; W. W. McGuire et al, (1982), J. Clin. Invest., 69, 543).
Elastase contributes also to the local inflammatory event going on in rheumatoid arthritis, e.g., the degradation of connective tissue constituents (K. Kleesiek et al (1985) in Neue Wege in der Entzundungsdiagnostik, PMN Elastase, p. 71-82).
The extracellular release of elastase after severe injuries or in diseases like septic shock, shock lung, etc. can be monitored routinely by means of enzyme immunoassays (S. Neumann and M. Jochum, (1984), p. 184-195 in Methods of Enzymatic Analysis, Bergmeyer (ed), Verlag Chemie, Weinheim).
In experimental models of sepsis and emphysema, synthetic elastase inhibitors (J. C. Powers, Am. Rev. Respir. Dis., (1983), 127, 554-558) and natural inhibitors from animals such as eglin C (H. P. Schnebli et al, (1985), Eur. J. Respir. Dis., 66, Suppl. 139 p. 66-70) have proven to be therapeutically useful. The application of a protease inhibitor of human origin would be preferable in order to avoid toxic side effects and especially allergic reactions when a prolonged therapy is indicated, e.g., in the treatment of .alpha..sub.1 -PI deficiency (emphysema). Since the human .alpha..sub.1 -PI is a glycoprotein of high molecular weight, its production in sufficient amounts by means of gene technology is not feasible in the near future.
The antileucoprotease or HUSI-I (H. Schiessler et al, 1978; and U. Seemuller et al, (1986), FEBS Letters 199, 43-48) has a MW of 14,000 Dalton and consists of two active domains, one directed against elastase, the other against trypsin. Hence this type of inhibitor has a relative low selectivity and is not a specific elastase inhibitor. Inhibition of trypsin or trypsin-like enzymes is normally not intended in the indications given above.
The human pancreas secretes the PSTI, a protease inhibitor of low MW (6.2 kd) which specifically inhibits trypsin, i.e., it possesses a high selectivity for one specific type of protease.
One advantage presented in this invention consists in substitution by recombinant DNA technology of only one (or a few) amino acids in the PSTI yielding PSTI variants which were shown to be highly effective protease inhibitors with high specificity for leukocyte elastase. Moreover, due to its relatively low MW the elastase PSTI derivative complex should pass through the kidney as well. Therefore, elimination of extracellularly released elastase will be highly efficient. The fact that PSTI is of human origin, taken together with its low MW lead applicants to expect that the clinical application of PSTI derivatives will be devoid of complications due to recognition of these substances as foreign proteins by the immune system.
Another essential advantage of PSTI compared to .alpha..sub.1 -PI and antileukoprotease is the insensitivity to oxidative inactivation during inflammatory processes whereby strongly oxidative agents are produced and released extracellularly. Consequently, compared to .alpha..sub.1 -PI or antileukoprotease, lower doses of the PSTI derivative should be sufficient to achieve a similar protective effect.